Nucleotides and nucleosides in the airway surface liquid (ASL) activate cell surface purinergic receptors that critically regulate airway epithelial fluid secretion and mucociliary clearance (MCC) functions. Nucleotide release rates and nucleotide/nucleoside levels in ASL are finely tuned to sustain MCC activities without promoting excessive fluid secretion or inflammation. However, the mechanisms of airway epithelial nucleotide release are incompletely understood. Studies in our lab have revealed that goblet and ciliated cells release nucleotides via exocytotic and conductive pathways, but there are important gaps in identifying/characterizing key components of these pathways. Our recent results provide insights into ATP uptake, storage, and release from goblet cell mucin granules. Our data suggest that the vesicular nucleotide transporter (VNUT) SLC17A9 regulates the uptake of ATP into mucin granules, and therefore contributes to mucin granule secretion-associated ATP release. Our results also suggest that the plasma membrane channel pannexin 1 mediates ATP release from ciliated cells, and that pannexin 1 activity is controlled by TRPV4 and Rho/ROCK activation. A plan to define the contribution of VNUT and pannexin 1 to nucleotide release from airway epithelia is outlined in this project by pursuing the following Specific Aims: (1) to test the hypothesis that VNUT contributes to mucin granule secretion-associated nucleotide release, and (2) to test the hypothesis that pannexin 1 channels mediate ATP release from ciliated cell-rich airway epithelia. We will utilize RNA interference techniques and gene targeting approaches in in vitro and in vivo models of airway epithelia, respectively, to assess (a) the extent to which VNUT contributes to nucleotide transport into mucin granules and to nucleotide release from mucin secreting epithelia, and (b) to define the role of pannexin 1 and upstream effectors in nucleotide release in the ASL. We will further explore the effect of VNUT and pannexin 1 gene deletions in the mucociliary and alveolar liquid clearance defects that have been attributed to unbalanced nucleotide release in asthma and viral infected lungs. Completion of these studies will establish molecular mechanism(s) for the physiologically important process of nucleotide release, and may delineate novel pharmacological strategies aimed to treat lung diseases associated with abnormal mucus clearance and inflammatory lung diseases.